1. Field of the Invention
The present invention relates to a novel D-lactic acid-producing strain and a use thereof. Specifically, the present invention relates to a method for preparing a D-lactic acid-producing strain including the steps of inhibiting L-lactate dehydrogenase (L-LDH) activity and introducing D-lactate dehydrogenase (D-LDH) activity in an L-lactic acid-producing strain, a modified D-lactic acid-producing strain prepared by the above method, and a method for producing D-lactic acid including the steps of culturing the strain and recovering D-lactic acid from the culture broth.
2. Description of the Related Art
Lactic acid has a wide range of industrial applications in foods, medicines, cosmetics, etc. In recent years, lactic acid has been utilized as a monomer of polylactic acid, and thus there has been a remarkable increase in demand for lactic acid.
Lactic acid can be produced by the chemical synthesis or the biological fermentation process using carbohydrates as a substrate. The latter is preferred from a commercial point of view because the chemical synthesis of lactic acid creates a problem of the cost increase caused by the gas price increase or environmental contamination. In addition, there are also problems of producing L-lactic acid in the form of a racemic mixture consisting of an equal amount of D-lactic acid and L-lactic acid. Unfortunately, the composition ratio of the D-lactic acid and the L-lactic acid cannot be controlled. When lactic acid in the form of a racemic mixture is used for preparing polylactic acid, an amorphous polymer with a low melting point is produced, thus an application of it is limited. On the other hand, the biological fermentation process using microorganisms makes it possible to selectively produce D- or L-lactic acid depending on the strain used. For example, microorganisms such as Lactobacillus sp., Bacillus sp., Rhizopus sp., Streptococcus sp., or Enterococcus sp. usually produce L-lactic acid. Microorganisms such as Leuconostoc sp. and Lactobacillus vulgaricus usually produce D-lactic acid. In particular, due to D-lactic acid is not metabolized in the body, D-lactic acid can be used as a biomaterial in the medical field and also used as an optically active herbicide via esterification and chlorination. It has been known that an optically active herbicide, can considerably improve its pharmaceutical effect and also has the same pharmaceutical effect with a lesser amount. For this reason, a demand for D-lactic acid has been increasing. In addition, sc-polylactic acid (stereocomplex-PLA) has a significantly higher melting point and thermal degradation temperature than the known polylactic acids. Therefore, it can be used as a high heat-resistant plastic material, resulting from a mixture of pure L-polylactic acid and pure D-polylactic acid. Consequently, a monomer of D-lactic acid is needed, and its demand has been gradually growing.
In producing such optically pure D-lactic acid, the biological fermentation process using enantioselective substrate specificity of a microbial enzyme is preferred. However, the wild-type, D-lactic acid-producing microorganisms generally found in nature, are still not suitable for industrial use regarding of optical purity or productivity. Examples of the D-lactic acid-producing microorganisms are Lb. plantarum, Lb. pentosus, fermentum, Lb delbrueckii, or the like. However, there are disadvantages that they are not able to produce lactic acid with high productivity and high yield, and 20˜40% of the lactic acid is L-lactic acid as an optical impurity. To overcome these disadvantages, attempts have been made to develop a variant producing high concentrations of lactic acid in a high glucose medium by inducing mutations in lactic acid-producing bacteria with treatment of EMS (ethyl methanesulfonate) (J. Industrial Microbiol, 11:23-28, 1992). As a result, the strain showing about a 4.8-fold higher productivity than a control group was selected, but its activity was reduced during long-term storage. Meanwhile, in the case of strain development using a variant, a yield-improved strain tends to show reduced productivity, whereas a productivity-improved strain tends to show reduced yield.
Based on the idea that strains for industrial lactic acid fermentation are generally L-lactic acid-producing microorganisms, and these microorganisms have mostly superior productivity and yield compared to D-lactic acid-producing microorganisms, the present inventors found that D-lactic acid can be produced in high yield by inactivating an L-lactate dehydrogenase (L-LDH)-encoding gene in a high L-lactic acid-producing microorganism and then introducing a heterogenous D-lactate dehydrogenase (D-LDH)-encoding gene thereto, thereby completing the present invention.